Brain samples were processed with antigen-retrieved citrate acid buffer (pH 6.0) and microwave for 30 min. The samples were permeabilized and blocked with PBS containing 5% goat serum and 0.3% Triton X-100 for 1 hour at room temperature. For BrdU labelling detection, sections were incubated with 2N HCl for 30 min at 37 °C and rinsed in 0.1 M borate buffer (pH 8.5) before blocking. For cell slides, the samples were fixed with 4% PFA at room temperature for 20 min and blocked with 5% goat serum. After blocking, the samples were incubated with primary antibodies and stained with fluorochrome conjugated secondary antibodies, counterstained the nucleus with DAPI and mounted with antifade medium (Dako). Cell image were obtained by regular confocal microscope (Zeiss LSM 800, Germany; Core facility in LSK Faculty of Medicine, HKU) and analyzed by Zeiss software.
For flow cytometry
After 48 hours of BYC and CO1 treatment, the C17.2 cells were incubated with the cultured medium adding 0.5 µM JC-1 (Invitrogen), 1 µM mitotracker green (Invitrogen) and 5 µM CellROX green. JC-1 was utilized for mitochondria membrane potential, mitotracker green was used for mitochondrial mass and cellROX probe was applied for intracellular ROS level detection. After 30 min staining, the cells were washed by PBS for 3 times and detached by trypsin. Flow cytometric analysis was conducted on FACS LSR II (BD Biosciences) and data were analyzed with FlowJo software (Treestar, Ashland).
For Western Blot
Proteins samples from brain tissue and cells were extracted by radioimmunoprecipitation assay buffer plus with 1% protease and phosphatase inhibitor cocktails (Cell Signaling Technology). Protein concentration was tested by BCA Protein Assay Kit (Thermo Fisher Scientific), separated by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and transferred on 0.45 µM polyvinylidene fluoride (PVDF) membranes. The membrane was blocked by 5% bovine serum albumin solution for 1 hour at room temperature and immunoblotted with primary antibodies following HRP-conjugated secondary antibodies. Immunoreactive bands on the membrane were revealed by chemiluminescent ECL select kit (GE Healthcare), visualized by Gel-Doc system (Bio-Rad) and analyzed by Image Lab software (Bio-Rad).